Formulation of Fast-Release Gastroretentive Solid Dispersion of Glibenclamide with Gelucire 50/13

Purpose: Fast-release gastroretentive solid dispersions of glibenclamide using gelucire were prepared to achieve improved bioavailability. Methods: Hot melt granulation technique was adopted to prepare solid dispersions (SDs) of glibenclamide in gelucire 50/13 and were compared with pure glibenclamide and physical mixtures of drug and gelucire using hot stage polarized microscopy, powder x-ray diffraction (PXRD), Fourier-transform infrared spectroscopy FTIR, bouyancy as well as by in vitro release and in vivo studies. Further aging studies were carried out for the samples. Results: PXRD showed that glibenclamide was present in SD in an amorphous form while FTIR spectroscopy revealed the presence of hydrogen bonding in the SDs. In vitro buoyancy was found for 11 h and there was improvement in solubility and dissolution rate for all test formulations. Formulations were found to follow Zero order kinetic. . During aging study, no decrease of in vitro drug dissolution was observed over 3-month period. Crystallinity in the SDs was observed following aging. A more pronounced lowering of blood glucose level in Wistar rats compared with the pure drug, suggests that the test formulations are superior. Conclusion: This study demonstrates the high potential of hot melt technique for obtaining stable fast-release gastroretentive solid dispersions of poorly water soluble drug using polyglycolized glycerides as carriers


INTRODUCTION
The gastro-residence time of orally administered dosage form is generally short due to rapid gastric emptying.Rapid gastrointestinal transit could result in incomplete drug release from orally administered dosage form above the absorption zone, leading to diminished efficacy [1].In order to increase the bioavailability of such drugs, the residence time of the orally administered dosage form in the upper gastrointestinal tract needs to be prolonged.The approaches to prolong gastroresidence time of pharmaceutical dosage forms include bioadhesive, mucoadhesive and density control delivery system [2][3][4].
Recently, much attention has been focused on the use of fats and fatty acid as carriers in drug delivery systems [5][6][7].Mahadik et al [8] demonstrated the use of amphiphilic lipid glyceryl monooleate for the design of floating matrix system.Gelucire is in the family of vehicles derived from mixtures of mono-, diand tri-glycerides with PEG esters of fatty acids.These are available with a range of properties depending on their hydrophiliclipophilic balance (HLB) value and melting point range (33 -65 ºC).These are used in the preparation of fast release and sustained release formulations.Gelucire containing only PEG esters are generally used in the preparation of fast release formulation.Owing to their extreme hydrophilicity and low density, Gelucire 50/13 may be considered an appropriate carrier for designing a fast release floating drug delivery system [9].Glibenclamide is a poorly soluble drug with possible content uniformity problems and dissolution rate-limited bioavailability, is given in a therapeutic dose of 5 -15 mg daily, has a half-life of around 10 h and exhibits low bioavailability.However, the bioavailability of the drug has been found to reduce further with conventional dosage forms probably due to the fact that passage of the single unit dosage form of the drug is faster than its release and most of the drug is released in the colon [10].Therefore, it is desirable to improve on earlier formulations by developing fast release gastroretentive solid dispersion system.Solubility enhancement of glibenclamide is an important aspect of formulation development.Although there is a plethora of reports of solubility improvement using different techniques, a comparative study of different solubilization approaches are few [11].Solubilization of poorly aqueous soluble drug forms an important activity in formulation process [12].Therefore, the objective of this study was to design and develop, with the aid of solubilizers, glibenclamide formulation with enhanced solubility using a solid dispersion approach.

EXPERIMENTAL Materials
Glibenclamide was received as a gift from Ranbaxy, Gurgaon, India.Gelucire 50/13 (semi-synthetic polyglycolized glycerides) was provided by Gattefosse, St.Priest, Cedex, France.All other materials and reagents used were of analytical grade.

Preparation of solid dispersions (SDs) and physical mixtures (PMs)
Solubility enhancement of glibenclamide (GLB) was achieved by dispersing the drug in molten Gelucire 50/13 at various ratios (1:1, 1:2, 1:4 and 1:10) [9].It was poured on aluminium foil, allowed to solidify in a covered Petri dish and then kept in a refrigerator for 3 h.The solid lump was passed through a fine mesh (150 µm) to obtain a fine powder formulation that was then placed in a calcium chloride desiccator for 48 h.The quantities of GLB, SDs and hydrophilic additives, namely, polyethylene glycol (PEG) 200, 400, 4000 and 6000 used are as shown in Table 1.Physical mixtures (PMs) of drug and Gelucire were prepared by triturating them for 15 min followed by sieving through a 150 µm mesh.

Evaluation of saturation solubility
The saturation solubility of GLB, PMs and SDs was evaluated by adding known excess amount of GLB formulation or pure GLB to 10 ml of 0.1M HCl (pH 1.2), stirred at 20 rpm in a water bath (25 ± 0.3 ºC) for 48 h, filtered, diluted suitably with 0.1M HCl (pH 1.2) and analyzed at 229 nm.

Hot stage polarized microscopy (HSPM)
About 2 mg of sample was placed on a glass slide and covered with cover slip.The slide was examined under optical microscope fitted with a hot stage, heated at a rate of 2 º C/min from room temperature to 50 ºC where it was held for 30 min, and then cooled to 40 ºC.It was held at this temperature for 30 min.Polarized light microscopy was applied for the detection of crystallinity [13].

Powder x-ray diffraction (PXRD)
X-ray diffraction studies on GLB, Gelucire 50/13, PMs and SDs were determined using a D8 Advance, Bruker AXS instrument with a nickel-filtered radiation.The samples were irradiated with monochromatized CuK (α) radiation (1.542 Å) and analyzed between 2 º and 50 º 2θ using a step scan mode (step width = 0.020 º (2θ), counting time = 0.5 s/step).Diffraction peak (d) intensities and 2 h values of the SDs patterns were compared to those of the pure materials in order to evaluate the physical form of GLB in the samples.

Fourier-transform infrared spectroscopy (FTIR)
FTIR spectra of the individual materials as well as SDs were obtained, after appropriate background subtraction, using FTIR-8400 spectrometer (Shimadzu, Japan).About 2 -3 mg of the sample was triturated with dry potassium bromide, compressed into disc and scanned from 4000 -400 cm −1 .

Evaluation of in-vitro buoyancy
In vitro bouyancy assessment was performed using a USP dissolution apparatus type II by placing the SD in 900 ml of 0.1M HCl (pH 1.2) at 37.0 ± 0.5 ºC and then agitated with a paddle at 75 rpm for 12 h.After agitation, the lipid particles that floated on the surface of the medium as well as those that settled at the bottom of the flask were removed separately.The proportion of floating particles was evaluated [14].
In vitro drug release studies USP type II (paddle) method was used with the aid of Electrolab dissolution tester (TDT06 N, India).The dissolution medium used was 900 mL of 0.1M HCl (pH 1.2) at 37 ± 0.5 °C and stirred at 75 rpm.An aliquot (5 ml) was withdrawn at predetermined time intervals for 4 h and replaced with same volume of fresh medium.The withdrawn sample was suitably diluted and analyzed using a spectrophotometer at 229 nm; the test was also carried out on a commercial brand of fast-release glibenclamide tablet (Betanase®, Cadila Healthcare Ltd, India).

Analysis of release kinetic and drug release mechanism
The release data obtained were treated according to zero-order, first-order, Higuchi [15] and Korsmeyer-Peppas's models [16] with the aid of PCP-Disso software (V3, Poona College of Pharmacy, Pune, India), in order to analyze the kinetics of drug release from the formulations.Higuchi (Eq 1) and Korsmeyer-Peppas (Eq 2) models were also applied to determine drug release mechanism.
where Q is the amount of drug released in time, t,, and K is the release constant.
where M t /M ∞ is the fraction of drug released in time, t, K is the structural and geometric constant, and n is the release exponent [17].

Effect of ageing
The SDs were stored at 30 ºC/65 % RH for 3 months and the effect of ageing on the SDs were studied by measuring their in vitro release as well as structural features using DSC and XRPD.

Evaluation of blood glucose
Blood glucose level (BGL) lowering studies of the SDs and pure GLBs were determined in streptozotocin (STZ)-induced diabetic Wistar rats of either sex weighing 150 -200 g.The animals were handled as per CPCSEA Guidelines of Good Laboratory Practice (GLP) [18].

Statistical analysis
Statistical analysis of the data was carried out by Student's t-test and one-way analysis of variance (ANOVA) at a significance level of p < 0.05 using SPSS 12.0 software (IBM).

Saturation solubility
The saturation solubility of GLB was 18.9 µg/ml while the enhanced saturated solubility obtained using drug:Gelucire (1:1) in physical mixture and solid dispersion was 27.23 and 44.39 µg/ml, respectively.Drug solubility increased in direct proportion to the proportion of Gelucire 50/13 in the preparations.Based on saturation solubility, ratio 1:10 showed enhanced solubility.

Hot stage polarized microscopy (HSPM)
HSPM of the SDs showed continuous melting from room temperature to 50 ºC and when it was cooled back to 40 ºC.Change in physical form was observed at different .The photomicrographs indicate that large crystals of pure GLB were reduced to small particle size when they came in close contact with the hydrophilic carrier.

X-ray diffraction
The x-ray diffractograms of pure GLB (Fig 2a)

In vitro buoyancy, and drug entrapment and release
In vitro release profiles of the GLB SDs formulation in simulated gastric (pH 1. ) data based on kinetic analysis using various release models are listed in Table 2.The formulations were best fitted to the zero-order release model, with r 2 close to one.Korsmeyer-Peppas n data also indicate that the drug release mechanism was non-Fickian case II diffusioncontrolled with n values ranging from 0.80 to 0.9221).The in vitro release data of optimized test GLB solid dispersion and commercial reference brand (Betanase® tablet) are indicated in Fig 3B .Release profiles of the two preparations were comparable.

Effect of ageing
When the GLB SDs were kept at 30 ºC / 65 %RH for 3 months, no change in in vitro drug dissolution was observed, compared with the initial release rate.

DISCUSSION
Hot melt granulation technique was selected to achieve solid dispersion, as it has been successfully utilized to increase the solubility of GLB [1].PEGs and Gelucire are among the several carriers that have been employed in preparing solid dispersions.
The purpose of the current study was to examine the solid-state properties of a solid dispersion system of GLB prepared using Gelucire 50/13 and various grades of PEGs at varying ratios.Intermolecular interaction such as hydrogen bonding between the amide of GLB and the oxygen of polyglycol chain (Gelucire) inducing a shift of N-H vibration to an extent that depends on the strength of interaction.The site of the interaction on Gelucire would probably have been C=O group, which would also affect N-H vibration.This observation agrees with the data generated from PXRD and FTIR studies.
Both HSPM and x-ray diffraction analysis revealed that GLB was in an amorphous state and uniformly distributed throughout matrix while FTIR spectroscopy revealed the possibility of H-bonding interaction in both PMs and SDs.
To achieve gastro-retention, the time needed for the initiation of floatation (floating lag time) was less than 3 min for all the SD formulations.However, maximum in vitro buoyancy was 11 h.SDs exhibited dramatically improvement in initial rate as well as extent of in vitro drug dissolution.Zero-order kinetics model fitted best for the formulations while the n exponent of the Korsmeyer-Peppas model indicate that erosion was the mechanism of drug release.Release of drug from hydrophilic minimatrices generally involves both pore diffusion and matrix erosion [5].Low density and hydrophilic property due to the higher HLB of Gelucire 50/13 facilitated gastroretension and drug dissolution.Gelucire 50/13 possesses surfactant and self-emulsifying property and is also used as meltable binder by melt granulation of poorly water soluble active substances [9].In contact with aqueous fluids, it forms a fine emulsion, solubilizes the active substance and hence increases its oral bioavailability.
Incorporation of GLB in the Gelucire 50/13 led to the formation of a solid dispersion system with increased dissolution rate of the drug due to improved wettability and the additional presence of a self-emulsifying compound, PEG.Moreover, it also increased the dispersability of the hydrophobic drug in the hydrophilic carrier during the process of solid dispersion formation.Thus, the increase in the dissolution of the drug can be attributable to improvement in wetting and to local solubilization by the excipients in the diffusion layer.The SDs exhibited higher burst release due probably to enhanced wettability of the drug particles, the emulsifying effect of carriers, significant reduction in particle size during SD formation and/or the inherently higher dissolution rate of soluble component of the SDs, which would pull along the more insoluble but finely mixed drug into the dissolution medium.Improved drug dissolution could also be attributed to the presence of the amorphous form of GLB, as indicated by the x-ray diffraction findings.
The increase in the release rate of the GLB SDs was also reflected in vivo by the greater reduction in blood glucose level in Wistar rats by GLB SDs, compared to pure GLB.

CONCLUSION
The present study demonstrates the high potential of hot melt-granulation technique for the production of solid dispersions of glibenclamide using polyglycolized glycerides as carriers.However, further studies are required to develop the formulation to industrial scale production.

Fig 1 :
Fig 1: Typical photomicrographs of solid dispersions (SDs) showing physical changes as temperature is varied temperatures as shown in the photomicrographs in Fig 1..The photomicrographs indicate that large crystals of pure GLB were reduced to small particle size when they came in close contact with the hydrophilic carrier.
had prominent diffraction peaks (d) equal to 8.035 º, 7.464 º, 4.649 º, 3.860 º, 2.933 º and 1.687 º, respectively on 2θ scale, which indicates its crystalline nature.The diffractograms for the formulations and Gelucire 50/13 (Fig 2b) indicate that GLB peaks decreased, suggesting its conversion from a crystalline to an amorphous state; diffractograms for the formulations physical mixtures and solid dispersions (Fig 2 (c) and (d)) indicate that GLB peaks decreased, suggesting its conversion from crystalline to an amorphous state; Gelucire 50/13 showed two prominent diffraction peaks (d) of 4.61 º and 3.81 º with the highest intensity on 2θ scale.The principal peaks of Gelucire 50/13 were present in both PMs and SDs with a lower intensity.The diffractogram of SDs showed absence of any trace of crystallinity, indicating the existence of amorphous GLB.
2) are shown in Fig 3A).In vitro buoyancy results indicate that the SD formulation remained floating for 11 h while drug entrapment efficiency was as high as 99.8 %.The regression coefficient (r 2

Table 2 :
Release kinetic data for GLB solid dispersions (SDs) based on various models Note: Formulation code as inTable 1; r 2 = regression coefficient.